The tuberous sclerosis complex (TSC) is an autosomal dominant, multisystem disorder that affects the brain and results from mutations in one of two genes, TSC1, encoding hamartin, and TSC2, encoding tuberin. Neurological complications of TSC are the most disabling and include epilepsy in over 70-80% of TSC patients, as well as autism and mental retardation in half of TSC patients. These neuropsychiatric abnormalities in TSC result from the effects of cortical tubers, the characteristic brain lesions of TSC, on brain function. Tubers are developmental abnormalities of cerebral cortical cytoarchitecture (a form of cortical dysplasia) characterized histologically by disorganized cortical lamination and cells with aberrant morphologies. The prominent abnormal cell types in tubers are dysplastic neurons (DN), giant cells (GC), and abnormal astrocytes. Tubers are epileptogenic and seizures in TSC patients are often refractory to medical management despite anticonvulsant polytherapy. Surgical resection of tubers may be necessary to achieve adequate seizure control. The number of tubers present in TSC patients seems to correlate with the onset and severity of mental retardation and autism in TSC patients. The broad goal of this grant proposal is to investigate how hamartin and tuberin mutations contribute to tuber formation using 3 experimental paradigms. We will determine whether tubers form as a result of a "second hit" somatic mutation or haploinsufficency using a high resolution analysis of TSC1 and TSC2 genes in single microdissected GCs, DNs, and astrocytes. Second, we define the expression of five candidate gene and protein families that are pivotal in normal corticogenesis including cell adhesion molecules, transcription factors, growth factors, and cytoskeletal elements in single DNs, GCs, and astrocytes and then relate these changes in expression to the mutational state of these cell types. Third, the expression of the five candidate gene and protein families will be determined during distinct epochs of cortical developmental in 3 transgenic mouse strains in which tuberin or hamartin have been completely or conditionally knocked out. These experiments provide a strategy to define the molecular mechanism of tuber formation as a direct consequence of TSC gene mutations and the downstream effects on gene expression within distinct populations of cells at defined developmental timepoints.